Spt23p and Mga2p of Saccharomyces cerevisiae are members of the highly conserved family of NF-kB like transcriptional regulators. Similar to their mammalian orthologues, proteins encoded by SPT23 and MGA2 undergo a unique limited proteasome-dependent event that generates the transcriptional competent polypeptides. In addition, the generated transcriptionally active polypeptides are held in a latent state outside of the nucleus and their nuclear mobilization is dependent on ubiquitination and the proteasome. A major difference between the yeast and NF-kB proteins is that Spt23p and Mga2p contain a carboxy-terminal transmembrane domain and are expressed as ER anchored proteins. Genetic studies have indicated that in addition to the proteasome, the highly conserved ubiquitin ligase RspSp and the ubiquitin binding Cdc48p- Npl4p-Ufd1 p segregase complex are required for the OLE1 -inducing function of Spt23p and Mga2p. In the case of Spt23p, RspSp is required for proteasome-dependent processing and recent studies indicate that this is mediated by a single RspSp interacting LPKY motif that is present with the carboxy-terminus of Spt23p. Curiously, RspSp and the LPKY motif is dispensable for Mga2p processing and recent published data suggests that RspSp facilitates Mga2p release by a mechanism that is distinct from Spt23p. The goal of experiments proposed here is to further define the various and poorly understood roles of the ubiquitin- proteasome pathway in Spt23p and Mga2p expression and activation. The Specific Aims of this proposal are 1) To perform a detailed structure-function based analysis of RspSp, Mga2p and Spt23p, 2) To determine the role of RspSp in release of transcriptionally competent Mga2p and Spt23p from the ER, and 3) To elucidate the mechanism of Mga2p90 and Spt23p90 generation by the proteasome. These studies will provide important new details relating to how the ubiquitin-proteasome pathway impinges on the activity and regulation of NF-kB like and membrane-localized proteins in eukaryotic cells.